Cellular origin of blocking factors from cultured spleen cells of tumor-bearing mice

Spleen cells from mice bearing methylcholanthrene-induced tumors were cultured for 2 days without further stimulation. Blocking factors were consistently detected in culture supernatants by their ability to suppress leukocyte adherence inhibition reactions between soluble tumor antigens and peritoneal cells of tumor-bearing mice. The blocking factors were specific for individual tumors. The cellular origin of these factors was investigated by depleting the spleen cell population of various cell types before culturing. The cells involved were removed by treatment with antibodies to certain membrane markers (Thy-1, Ly-2, Ia, I-J) but not by anti-Ly-1 antibodies. Removal of adherent cells also prevented production of blocking factors, which was restored by reconstitution with syngeneic but not allogeneic cells from normal mice. The normal reconstituting cells were shown to bear Ia, but not I-J or IgM. This indicates that blocking factors (previously shown to have I-J determinants in their molecules) originate from suppressor T lymphocytes (Thy-1+, Ly-1-2+, I-J+), with macrophages (I-J-, Ia+) in the role of accessory cells

Further characterization of the cells involved in leukocyte adherence inhibition with murine tumor extracts

Peritoneal cells from tumor-bearing CBA mice were depleted of either T lymphocytes (by treatment with anti-Thy-1.2 antiserum), lymphocytes with the Ly-1 marker (by anti-Ly-1.1 monoclonal antibody), or macrophages (by an adherence method). Depleted populations were compared with control populations for their ability to react with the corresponding tumor extract in the leukocyte adherence inhibition (LAI) assay. Direct and indirect assays were used, with emphasis on the latter which depends on the formation of a soluble mediator, leukocyte adherence inhibition factor (LAIF). Both Moloney sarcoma virus-induced tumors and transplanted methylcholanthrene-induced tumors were employed and gave similar results. LAI reactivity depended on lymphocytes with the Thy-1 and Ly-1 markers, and on macrophages (syngeneic or allogeneic). These T lymphocytes were shown to be the source of LAIF, the macrophages being required as accessory cells. T lymphocytes were also necessary in the LAIF-responsive indicator cell population

A two-chain tumour-related suppressor factor specific for a sequence antigen

Suppressor factors in the serum of CBA mice bearing transplanted methylcholanthrene-induced tumours suppressed the leukocyte adherence inhibition reaction between tumour-sensitized peritoneal cells and a sequenced antigen, myelin basic protein (MBP). Chromatography on Sephacryl S-200 columns demonstrated suppressive activity in low molecular weight fractions (40-50 kDa) derived from whole serum. The factors were absorbed from serum by MBP, but not by lysozyme, coupled to Sepharose gel. Cleavage with sodium dodecyl sulphate (SDS) and absorption with either MBP or anti-I-J antibody on Sepharose gels yielded inactive supernatants that regained suppressive activity when combined. Elution of these gels with acidic buffer yielded eluates that were inactive alone but suppressive when combined. These results indicate that the tumour-related suppressor factors consist of two chains separable by detergent action; one chain is antigen (MBP)-binding and the other contains an I-J determinant